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Molecular clocks mediate a daily transcriptional schedule that maintains normal tissue physiology. Glucocorticoids synchronise peripheral clocks with the master clock in the suprachiasmatic nucleus in response to ambient light. Evidence from humans and mice suggests that arrhythmic glucocorticoids induce non-dipping blood pressure and vascular dysfunction. The mechanisms of this are poorly understood. I hypothesized that arrhythmic activation of the glucocorticoid receptor attenuates the molecular clock mechanism in the renal artery, a major component of blood pressure control, dysregulating its normal function. I tested this hypothesis in a mouse model with clamped corticosterone rhythm, first by defining the circadian transcriptome of the renal artery and then by measuring the day-night circadian differences in renal artery physiology RNA sequencing revealed the circadian transcriptome of renal arteries under control conditions and with arrhythmic glucocorticoids. In the control group 465 (3%) transcripts out of 14425 protein-coding genes were rhythmic. 195 of those genes lost their rhythm under arrhythmic corticosterone and were related to pathways including circadian transcription, TGFβ signalling and extracellular matrix. Paradoxically, several transcripts in the arrhythmic corticosterone group gained rhythmicity and were indicative of mitochondrial respiratory chain and oxidative phosphorylation pathways. In parallel, messenger RNA (mRNA) levels of all elements of the core clock machinery were significantly dysregulated. Additionally, transcripts of mediators of the arterial vasomotor machinery were significantly changed in the arrhythmic corticosterone mice compared to controls, while levels of the glucocorticoid receptor (GR) were significantly downregulated. To investigate underlying mechanisms of glucocorticoid control of renal artery function I followed two approaches. First, I compared the day/night reactivity to vasoactive stimuli in isolated renal arteries from control and arrhythmic corticosterone mice. Arteries from control mice displayed greater endothelial-dependent (via acetylcholine) and -independent vasodilation (via nitric oxide donor sodium nitroprusside) was higher during the dark phase (active period) compared with the light phase (inactive phase). In the arrhythmic corticosterone group time of day variability in response to both vasodilators was attenuated. Second, to interrogate the role of glucocorticoid signalling within the smooth muscle in circadian vascular reactivity, renal arteries from mice lacking the glucocorticoid receptor in the smooth muscle (SMGRKO) were used. Time of day dependent vascular reactivity to endothelial-independent vasodilation seen in the controls, was attenuated in the SMGRKO animals. I speculate that temporal misalignment of the vascular molecular circadian machinery, due to arrhythmic glucocorticoid signalling in the arrhythmic corticosterone and SMGRKO mice, may cause subsequent temporal changes in gene expression and have a direct effect on dilatory capacity. In summary I found a robust effect of arrhythmic glucocorticoid signalling both at the transcriptional and functional level of the renal artery circadian physiology. Arrhythmic glucocorticoids may achieve this via several potential pathways including the ability of the renal artery to respond to nitric oxide. Together, this novel study extends our understanding of circadian vascular physiology and underlines the impact of glucocorticoid rhythm on temporal changes in gene expression. This may be clinically relevant in the pathogenesis of vascular dysfunction associated with elevated glucocorticoids in metabolic syndrome and chronic stress.

Circadian rhythms are self-sustained biological oscillations with a period of approximately 24 hours. They are observed across all levels of biological scale and organise physiology to accommodate the different environmental and energetic demands of day and night. In mammalian cells, the mechanistic target of rapamycin complex (mTORC) activity exhibits daily rhythms both in vivo and in cultured cells. Circadian rhythms are sensitive to changes in mTORC activity, moreover, many circadian-regulated process are directly or indirectly sensitive to changes in mTORC activity e.g., protein synthesis and macromolecular crowding. In my research I have sought to gain insight into the nature of the interaction between circadian regulation and mTORC signalling, in vitro and in vivo. My lab previously observed reciprocal circadian rhythms in the abundance of soluble proteins and ions, which function to maintain cellular osmotic homeostasis and are dependent on mTORC activity. In the first part of this thesis, I explore the consequences of osmotic perturbation on circadian rhythms, and find that acute osmotic perturbations modulate circadian rhythms via mTORC. Next, I sought to understand the generation and function of cell-autonomous soluble protein, and functionally characterise them by quantitative proteomics and phosphoproteomics. I find that, in both cultured cells and in mouse liver, soluble protein rhythms are modulated by mTORC activity. Through pharmacological inhibition of mTORC1 and 2 I explore how circadian regulation of physiological outputs, such as wound healing, is mediated by mTORC activity, as well as the effect of mTORC inhibition on circadian timing in vitro and in vivo. Taken together, this work points to a model where rhythmic mTORC may be sufficient to modulate signalling to and from the circadian timekeeping machinery to drive most rhythmic physiological outputs, both in vitro and in vivo.

Circadian rhythms orchestrate physiology across all kingdoms of life to anticipate the differing demands of day and night. Circadian timekeeping has a cell-autonomous basis, amenable to study in cultured mammalian cells, where daily rhythms have been shown to affect most biological processes. In various models, rhythms in protein abundance of 10-20% of the proteome have been reported. However, regulation of these rhythms is not well understood, as most of them are not accounted for at the transcript or even ribosomal occupancy level. It is also unclear how circadian rhythmicity in protein translation and abundance affects protein homeostasis, maintenance of which is crucial for cell viability. In this thesis, I report the investigation of post-transcriptional temporal control of protein abundance, and the interplay between protein homeostasis and the circadian clock. I was particularly interested in proteasomal degradation, as it has been implicated in a variety of circadian model systems, and is likely to have a conserved role both feeding into and being regulated by cellular timekeeping. I provide data in support of this in the first results chapter, showing that proteasome activity is essential for temporal organisation of circadian transcriptional networks, and that cellular proteasome activity is circadian-regulated. In the subsequent chapter, using various protein labelling strategies, I describe circadian variation in global protein degradation. A rhythm in global protein synthesis has been reported previously, but I found this occurs in-phase with the rhythm in degradation. It suggests that protein turnover and proteome renewal is temporally consolidated. Data from cells deficient in circadian transcriptional feedback, where proteostasis is perturbed and more proteins have rhythms in abundance, supported the idea that circadian rhythms could function to maintain protein homeostasis, rather than to generate protein abundance rhythms. To explore this new model proteome-wide, I then optimised and performed a complex dynamic proteomics study, using a cutting-edge pulsed SILAC-TMT methodology to measure protein synthesis, degradation and abundance across circadian time. This provided a detailed picture of temporal protein regulation, and, among other things, showed that protein synthesis varies more over time than does steady-state abundance, in terms of number of rhythmic proteins and relative amplitude. Finally, I also looked into the consequences of rhythms in protein turnover. I predicted that at times of higher turnover, cells would be more sensitive to proteotoxic stress. This appeared true at the level of stress response markers and protein aggregation, as well as cell viability after a proteostasis challenge. Time-of-day variation in turnover was also observed in animal tissues, as was evidence for time-of-day variation in the effect of bortezomib, a clinically relevant proteasomal inhibitor. Altogether, this work provides new insights into temporal control of protein homeostasis. It points to a novel model where circadian timekeeping acts to promote proteostasis and to co-ordinate proteome renewal, which would be advantageous in terms of bioenergetic efficiency and osmotic homeostasis.

The hair follicle (HF) is a human mini-organ that autonomously cycles between phases of growth (anagen), regression (catagen) and relative quiescence (telogen). Whilst many molecular controls are now appreciated to influence hair cycle, what ultimately choreographs the switch between each cycle stage is yet to be elucidated. With the increasing link between molecular clock activities in controlling local tissue physiology, we began by studying the hypothesis that the human HF has a functional molecular clock. Utilising human HF organ culture, qRT-PCR and immunofluorescence we found that the HF does indeed have oscillating clock gene expression over 24 and 48 hours in situ, separate from the suprachiasmatic-nucleus. Moreover, core clock proteins BMAL1 and PER1 are expressed in the human HF with PER1 increasing as HFs enter catagen. Next utilising siRNA mediated gene knock-down of either BMAL1 or PER1 in situ, we were able to show that silencing either clock gene leads to anagen prolongation in cultured HFs, demonstrating that the molecular clock modulates the human hair cycle, namely the anagen-catagen switch in situ. As human pigmentation is tightly coupled to the hair cycle and both human HFs and epidermal melanocytes express clock genes/proteins, this led us to investigate the hypothesis that the molecular clock modulates human pigmentation. By silencing BMAL1 or PER1 in HFs an increase in melanin content (Masson-Fontana) was observed in a hair-cycle independent manner. Furthermore, tyrosinase expression/activity as well as TYRP1 and 2 expression, gp100 protein expression, melanocyte dendricity and the number of HF melanocytes were all significantly increased in BMAL1 and/or PER1-silenced HFs. Mechanistically, BMAL1 knockdown reduced PER1 transcription, and PER1 silencing was found to induce phosphorylation of the master regulator of melanogenesis, MITF, thus stimulating human melanogenesis and melanocyte activity. This provides the first evidence that the peripheral molecular clock influences human pigmentation. Finally, the thyroid hormone (T4) has strong links with peripheral clock activity and has been shown to prolong anagen and increase human HF pigmentation. Moreover, T4 is a commonly prescribed treatment for thyroid disorder. As such, we investigated the hypothesis that T4 influences HF clock gene activity. It was observed that transient T4 treatment reduces the amplitude of clock gene oscillations whilst circadian rhythmicity is maintained. Conversely with longer term treatment clock gene activity was significantly increased compared to a scrambled oligo-control. Here we have demonstrated that the human HF has peripheral molecular clock activity which influences the human hair cycle and pigmentation. Finally we were able to uncover a potential novel target, T4, whose pulsatile administration may potentially be used to treat not only hair growth and pigmentation disorders but may be able to modulate circadian activity in peripheral tissues and treat clock-related disease.

In mammals, circadian rhythms are driven by a cell-autonomous core-clock mechanism consisting of over a dozen core-clock genes forming transcription-translation feedback loops. The core-clock mechanism also drives the rhythmic expression of downstream genes called clock-controlled genes, which are thought to be important for driving rhythmic biochemical and physiological processes. Mathematical models predict that for a gene to be rhythmically expressed, synthesis, degradation, or a combination of the two must be rhythmic. The purpose of this project was to investigate the contribution of synthesis and degradation of RNA to rhythmic gene expression. To systematically understand the contribution of synthesis, degradation, and other RNA dynamics to rhythmic gene expression, I used metabolic labeling and a novel computational pipeline to analyze transcriptomic data in synchronized NIH3T3 cells. I identified 685 rhythmically expressed RNAs with a period of 24-hour in my dataset, of those 389 were rhythmically synthesized and 24 were rhythmically degraded. Low amplitude degradation rhythms were detected more broadly in the 685 rhythmically expressed RNAs, but these were not statistically significant. Although synthesis was the primary driver of rhythmic 24-hour RNA expression, core-clock gene RNAs were regulated by both synthesis and degradation, presumably to sustain high amplitude of rhythmic expression. I also identified rhythmic RNA expression with a period of 12 and 8 hours; interestingly, degradation primarily drove rhythmic expression of these RNAs. Overall this dissertation revealed RNA dynamics that drive rhythmic gene expression. This will provide insights into how diverse circadian clock mechanisms ultimately drive tissue-specific rhythmic gene expression.

Cyanobacteria are photosynthetic microbes that have shaped the very environment of the world we live in over several billion of years. This is, in large part, because of their ability to perform the chemically exclusive processes of photosynthesis and nitrogen fixation. To segregate these processes temporally, they have evolved an elegant and complex circadian clock that aligns their physiology with the solar day to maximize biological fitness. This clock keeps time through the action of a biochemical oscillator comprising just three proteins: KaiA, KaiB and KaiC, that, along with ATP, can recapitulate a 24-h pacemaking activity in vitro. This process is achieved by a negative feedback loop akin to a biochemical game of Rock, Paper, Scissors, whereby the phosphorylation state of the hexameric ATPase KaiC is controlled by the nucleotide exchange factor KaiA, the ability of KaiA to stimulate KaiC repression is controlled by the metamorphic protein KaiB, and the ability of KaiB to inactivate KaiA is controlled by KaiC phosphorylation state. This creates a repetitive biochemical cycle that takes just bout 24 hours to complete. Additionally, the output proteins SasA and CikA interact with the clock in various phases of the biochemical oscillation that influence their ability to activate the master circadian transcription factor RpaA and orchestrate circdain gene expression throughout the cyanobacterial cell.This thesis focuses on the relationship between KaiC and KaiB. In particular, on positive cooperativity that increases KaiB’s affinity for KaiC, or in other words, the process by which initial binding of KaiB to the KaiC hexamer enables more efficient recruitment of KaiB to the remaining 5 binding sites. While this effect is well documented when considering KaiB and KaiC on their own, in Chapter 2 we expand this concept in the in the context of the entire reconstituted clock system including output pathways that link biochemical oscillation to DNA binding by the transcription factor RpaA. In doing so, we uncovered the unexpected result that SasA expands the range of permissive KaiB concentrations for biochemical oscillation to occur. I showed that SasA does this by binding to KaiC analogously to how KaiB does, and then recruiting additional KaiB molecules through positive heterotropic cooperativity. Integrating a novel crystal structure of the interacting domains of KaiC and SasA with existing crystal structures of the KaiC hexamer, I identified SasA mutations that abrogate heterotropic cooperativity but have only minor effects on the output signaling function of SasA. Remarkably, cyanobacteria bearing these mutations have defective circadian rhythms, demonstrating that SasA’s ability to bolster KaiB recruitment is an evolved aspect of biochemical oscillation.Chapter 3 describes our structural analysis of phosphomimetic variants of KaiC that differ in their ability to bind KaiB. Because crystallography has failed to identify the structural basis of this discrimination previously, we employed cryo-electron microscopy to analyze KaiC particles as a structural ensemble frozen in ice. Each KaiC protomer is composed of two ATPase domains, termed CI and CII. The phase-determining phosphosites are located on CII, and KaiB binds to KaiC over 70 Å away to the ADP-bound form of CI. Our data corroborated previous studies that daytime KaiC, which does not bind KaiB, loses interactions amongst the CII domain protomers within the hexamer, while maintaining a hexameric CI domain. Additionally, we obtained a relatively high resolution (3.2 Å) structure of a compressed form of KaiC where the CII domain breaks into a split washer with 2-fold symmetry, causing two of the CII domain subunits to interact more tightly with their respective CI domains. Using mutagenesis and various functional assays, I identified an allosteric conduit that connects the ATPase domains of the CI and CII domains of KaiC in both intra-protomer and inter-protomer contexts. Importantly, I trace these interactions back to cooperativity in KaiB association, with mutants along the pathway disrupting both KaiB affinity and cooperativity. Furthermore, I link KaiB cooperativity to ATP hydrolysis in the CI domain by identifying a key residue that senses CI nucleotide state. This residue is dispensable for both ATP hydrolysis as well as KaiB association, but is critical for both KaiB cooperativity and in vivo circadian rhythms. This, along with additional nucleotide dependence studies reported in Chapter 4, suggests that allosteric control of cooperativity through the CI active site is the structural basis for restriction of KaiB association to the nighttime KaiC phosphostates.Finally, in the latter half of Chapter 4 I summarize experiments that used the solvatocromatic dye Sypro Orange to detect changes in KaiC structure as a function of temperature and mutagenesis. I describe our preliminary efforts to build on these results by identifying additional solvatochromatic dyes that can leverage this effect to report continuously on the phase of biochemical oscillation by discriminate binding to different KaiC phosphostates at constant temperature.

Biological rhythms are ubiquitous in nature. Examples include daily cycles of rest and activity observed among most living organisms and annual cycles of hibernation and migration observed among animals and birds. However, the timekeeping mechanisms that underlie the vast majority of biological rhythms are not well understood. I chose the molting cycle of C. elegans as a scientific model to study this problem. C. elegans molt 4 times at regular time intervals. Each life stage of the animal consists of the intermolt, a 6 – 8 h period of feeding and locomotion, which is followed by the molt, and a 2 h period of sleep-like quiescence and remodeling of the exoskeleton. I found that transcriptional–post-transcriptional feedback loops among the conserved nuclear hormone receptor NHR-23 and the let-7 family of miRNAs govern both the pace of the molting cycle and the total number of molts. The negative feedback loop acts within a genetic oscillator – the molting cycle timer – to schedule the timing of reiterative molts and within an hourglass timer to schedule the extinction of the molting cycle in adulthood. Knockdown of nhr-23 slowed down the pace of the cycle, whereas forced expression of nhr-23 or inactivation of let-7s accelerated the pace of the cycle. Interactions between NHR-23 and let-7s drive the cyclical expression of nhr-23, let-7s and scores of downstream targets necessary for the process of molting. I also found that the oscillatory expression of nhr-23 is dampened in regular increments over the course of larval development; the dampening was dependent upon the activity of let-7s. Moreover, forced expression of nhr-23 was sufficient to trigger extra molts in sexually mature animals. I propose that the incremental dampening brings the levels of nhr-23 below a critical threshold, preventing entry into another molt. Both the core components and cis-regulatory elements of this timekeeping mechanism are conserved from nematodes to humans. Going further, I identified additional genes and molecules that either act upstream of NHR-23 and let-7s or in independent pathways to control the number of molts in C. elegans. An as-yet-unidentified hormone is thought to control molting by acting as a ligand for NHR-23. Consistent with this model, I show that dietary supplementation of cholesterol is necessary for forced expression of NHR-23 to trigger extra molts. Thus, cholesterol or a cholesterol-derivative might act as a native ligand for NHR-23. Using a reverse genetics approach, I also uncovered genes that might act in pathways for synthesis of the ligand. My findings might lead to discovery of a hormone that promotes molting in C. elegans—a long-standing question in the field. Further examination of how the molecular and genetic interactions uncovered in this thesis regulate the molting cycle could lead to a better understanding of developmental clocks, as well as the circadian clock and related sleep, metabolic and psychiatric disorders.

Chronobiological aspects controlled by CLOCK genes may influence obesity incidence. Although there are studies that show an association between the expression of these genes and energy intake, waist circumference or abdominal obesity phenotypes, interactions with appetite have been insufficiently investigated in relation to chrononutrition. The objective was to identify interactions between CLOCK genetic variants involved in appetite status. A total of 442 subjects (329 women, 113 men; aged 18 to 65 years) were recruited. Anthropometric, dietary and lifestyle data were collected by trained nutritionists. Participants were classified according to their appetite feelings with a Likert scale. Multiple linear regression models were used to examine associations of the type genotype x appetite status on adiposity-related variables. p values were corrected by the Bonferroni method. A significant influence was found concerning the effects of appetite on waist circumference with respect to rs3749474 CLOCK polymorphism (p < 0.001). An additive model analysis (adjusted by age, gender, exercise and energy intake) showed that risk allele carriers, increased the waist circumference around 14 cm (β = 14.1, CI = 6.3–22.0) by each increment in the level of appetite. The effects of appetite on waist circumference may be partly modulated by the rs3749474 CLOCK polymorphism.

My experiment concerned the influence of chronobiological (circadian) rhythm on fatigue, effort, and cardiovascular (CV) response. It evaluated responses of morning people (Larks) presented an easy or difficult recognition memory task at a time congruent or incongruent with their rhythm. Based on an extension of a conceptual analysis of fatigue influence, my central prediction was that circadian rhythm would combine interactionally with task difficulty to determine effort and associated CV responses. Specifically, effort and associated CV responses were expected to be (1) positively correspondent to task difficulty in the morning (stronger where difficulty is high), but (2) negatively correspondent to difficulty in the evening (stronger where difficulty is low). Preliminary results showed concerning gender effects on difficulty appraisal of the task, thus we examined women and men's data separately. CV findings for women were broadly, but not completely, consistent with predictions. Analyses revealed no group differences in CV response for Lark men.

La consideración de la estructura de sistemas, de acontecimientos relacionales y de ciclos temporales observados en los seres vivos siempre ha sido una fuente de inspiración para el hombre. La naturaleza, como sistema abierto nos ofrece la oportunidad de aprender tanto de los resultados como de sus procesos. La construcción de nuevos paradigmas inspirados en la naturaleza será el hilo argumental del artículo: la naturaleza como material de construcción de nuevos modelos de pensamiento y de organización dentro del campo de la arquitectura y de la ciudad.